John W. Elrod

Extracellular matrix, junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation.

Vascular endothelial permeability is maintained by the regulated apposition of adherens and tight junctional proteins whose organization is controlled by several pharmacological and physiological mediators. Endothelial permeability changes are associated with: (1) the spatial redistribution of surface cadherins and occludin, (2) stabilization of focal adhesive bonds and (3) the progressive activation of matrix metalloproteinases (MMPs). In response to peroxide, histamine and EDTA, endothelial cells sequester VE‐cadherin and alter its cytoskeletal binding. Simultaneously, these mediators enhance focal adhesion to the substratum. Oxidants, cytokines and pharmacological mediators also trigger the activation of matrix metalloproteinases (MMPs) in a cytoskeleton and tyrosine phosphorylation dependent manner to degrade occludin, a well‐characterized tight junction element. These related in vitro phenomena appear to co‐operate during inflammation, to increase endothelial permeability, structurally stabilize cells while also remodelling cell junctions and substratum.

Troglitazone, a PPAR-γ activator prevents endothelial cell adhesion molecule expression and lymphocyte adhesion mediated by TNF-α

BackgroundCytokine mediated induction of the mucosal addressin cell adhesion molecule-1(MAdCAM-1) expression is associated with the onset and progression of inflammatory bowel disease (IBD).ResultsUsing western blotting and cell-based ELISA, we show in this study that troglitazone, an activator of the peroxisome proliferator-activated receptor-γ (PPAR-γ), widely used in the treatment of diabetes, has as well recently been highlighted as protective in models of inflammation and cancer. We found that troglitazone (10–40 μM), significantly reduced the TNF-α (1 ng/ml) mediated induction of endothelial MAdCAM-1 in a dose-dependent manner, achieving a 34.7% to 98.4% reduction in induced MAdCAM-1. Trogliazone (20μM) reduced TNF-α induced VCAM-1, ICAM-1 and E-selectin expression. Moreover, troglitazone significantly reduced α4β7-integrin dependent lymphocyte adhesion to TNF-α cultured endothelial cells.ConclusionsThese results suggest that PPAR-γ agonists like troglitazone may be useful in the clinical treatment of IBD.

Gamma knife irradiation increases cerebral endothelial expression of intercellular adhesion molecule 1 and E-selectin.

OBJECTIVEAlterations in multiple functions of the microvasculature occur in response to gamma irradiation and are thought to contribute to radiation-induced end organ damage by inducing inflammatory responses, particularly leukocyte infiltration into the affected area. Endothelial cell adhesion molecules (ECAMs) mediate leukocyte adhesion and migration. Here, we validate a method to study the effect of Leksell gamma knife stereotactic radiosurgery on the expression of ECAMs on human cerebral endothelium at 0, 24, 48, and 72 hours after irradiation. METHODSA human brain endothelial cell line (IHEC) was cultured on 12-mm coverslips and subjected to 50 Gy of collimated gamma irradiation with the Leksell gamma knife (Elekta Instruments, Inc., Atlanta, GA). Lactate dehydrogenase release was measured at 24, 48, and 72 hours after irradiation and caspase-3 at 24, 48, 72, 96, and 120 hours. ECAM expression was measured at postirradiation intervals of 0, 24, 48, and 72 hours by cell enzyme-linked immunoabsorbent assay. We used a cell irradiator composed of two chambers. The upper chamber holds the coverslips firmly in place while they are immersed in media. The lower chamber is connected to a peristaltic pump, which pumps water into the chamber and maintains the media in the upper chamber at 37°C through convection. RESULTSNone of the ECAMs tested was significantly elevated compared with the control basally. Twenty-four hours after irradiation, intercellular adhesion molecule 1 was significantly elevated on brain endothelial cells but there was no significant elevation of E-selectin. Vascular cell adhesion molecule 1 was increased slightly but not significantly and decreased at 48 hours. At 72 hours, E-selectin expression was significantly increased; intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were not altered relative to sham controls. CONCLUSIONIncreased ECAM expression and lactate dehydrogenase release support the idea that the cerebral microvasculature undergoes an inflammatory response after Leksell gamma knife stereotactic radiosurgery.

Reciprocal Regulation of Endothelial Substrate Adhesion and Barrier Function

Objective: To examine how cell‐substrate adhesion is regulated during barrier changes produced by exposure to inflammatory mediators.

Identification of an occludin cell adhesion recognition sequence.

The molecular mechanisms by which the tight junction integral membrane protein, occludin promotes cell adhesion and establishes an endothelial monolayer permeability barrier have not been elucidated. In particular, the amino acid sequences of the occludin cell adhesion recognition (CAR) sites have not been determined. Here we demonstrate that a cyclic peptide containing the sequence LYHY, which is found in the second extracellular domain of occludins in all mammalian species, inhibits the establishment of endothelial cell barriers in vitro and in vivo. This cyclic peptide also prevents the aggregation of fibroblasts stably transfected with cDNA encoding occludin. The data suggest that the LYHY motif is an occludin CAR sequence.

MAdCAM‐1 expression and regulation in murine colonic endothelial cells in vitro

Background: Although the mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) is associated with the etiology of inflammatory bowel diseases, few studies have directly examined MAdCAM‐1 using microvascular endothelium derived from the colon. This study measured the expression of MAdCAM‐1 in a novel colon endothelial line MJC‐1, as well as MAdCAM‐1 regulation and function in vitro. Methods: We cloned microvascular endothelial cells from primary colon cultures using ImmortoMice mice (whose cells express a temperature‐sensitive SV40 large T antigen, H‐2Kb‐tsA58 mice). Expression of MAdCAM‐1 after stimulation with cytokines [tumor necrosis factor (TNF)‐&agr;, interleukin (IL)‐1&bgr;, or interferon (IFN)‐&ggr;] was determined by Western blotting. Signal paths regulating MAdCAM‐1 expression were examined using pharmacological blockers before cytokines. We also examined lymphocyte adhesion using lymphocytes that constitutively express &agr;4&bgr;7 integrin. Results: TNF‐&agr; induced MAdCAM‐1 in a dose‐dependent manner by 24 hours. MAdCAM‐1 induction was protein kinase C, tyrosine kinase, p38 mitogen activated protein kinase, and nuclear‐factor kappa‐B/poly adenosine diphosphate ribose polymerase dependent. Lymphocyte adhesion was increased 2.6‐fold after TNF‐&agr; stimulation and was inhibited by anti‐MAdCAM‐1 antibody before treatment (P < 0.05 control versus TNF‐&agr;). Conclusions: In vitro, MAdCAM‐1 can be induced on colon endothelial cells by TNF‐&agr; stimulation and may represent a useful model to study microvascular injury in the large intestine.

DSS‐induced colitis is exacerbated in STAT‐6 knockout mice

Background: Several transcription factors have been proposed to regulate IBD including the signal transducer and activator of transcription‐6 (STAT‐6). Methods: The role of STAT‐6 was examined in the 5% dextran sulfate sodium (DSS)‐induced murine model of colitis using STAT‐6−/− and wildtype mice. Results: The disease activity index (DAI) revealed a significant increase in DAI in STAT‐6−/− mice over STAT‐6+/+ mice given DSS. Both STAT‐6−/− and wildtype mice displayed severe inflammation and crypt damage. Additionally, STAT‐6−/− mice showed significant injury to the proximal colon compared with their littermate controls. Furthermore, STAT‐6−/− mice receiving DSS had dramatically higher levels of serum nitrite/nitrate than all other groups. STAT‐6−/− animals also displayed higher levels of inteferon‐&ggr; than wildtype mice. Conclusions: Because STAT‐6 has been reported to regulate the expression and activity of inducible NO synthase (iNOS), our data suggest that, in DSS colitis, STAT‐6 may modulate iNOS, to limit NO formation and control the extent of inflammation in the colon. We conclude that STAT‐6 may normally play an important regulatory role in the pathogenesis of inflammatory bowel disease, possibly through modulation of iNOS and interferon‐&ggr;.

Roles of Leukocyte and Immune Cell Junctional Proteins

The restricted expression of E‐cadherin on dendritic cells and intraepithelial lymphocytes has been described as a structural/adhesive system that helps to retain and integrate these cells within mucosal and dermal tissues. The activation of these cells downregulates expression of cadherins, and contributes to cell redistribution and tissue homing. It has recently been reported that lymphocytes and other leukocytes express cadherins, as well as occludin, a tight junctional component, in response to several types of stimuli. This suggests that mobilization of adherens and tight junction proteins in leukocytes may facilitate interactions of leukocytes with epithelial, endothelial, and interstitial cells that express these proteins and support homophilic adhesion. The conditions and patterns of synthesis of these adhesion molecules, in antigen‐presenting cells and leukocytes, indicate that the expression of junction proteins may play roles in normal and pathological leukocyte traffic.